Methods and compositions for the introduction of molecules into cells

ABSTRACT

The present invention is directed to the introduction of molecules, including nucleic acids, carbohydrates, plant growth regulators and peptides into cells and tissues. The present invention is also directed to media and methods for enhancing embryogenic callus production of elite lines of soybean.

BACKGROUND OF THE INVENTION

[0001] The present invention is directed to a method for theintroduction of molecules into cells, including but not limited tobacterial and plant cells. The molecules which are introduced by themethod of the invention include, without limitation, nucleic acids,carbohydrates, plant growth regulators and peptides. The method of theinvention is further directed to the transformation of bacteria andplant cells and tissues and to the resulting transformed cells andtissues. The present invention is also directed to a method and mediumfor initiating more rapid and uniform growth of embryogenic callus,specifically the growth of soybean embryogenic callus.

[0002] The publications, patents and other materials used herein toilluminate the background of the invention, and in particular cases, toprovide additional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

[0003] Delivery of Molecules to Cells

[0004] Small and large molecules can be efficiently delivered to cellswithout cell walls by electric pulsing (Dagher et al., 1991),electroporation (Fromm et al., 1986) or through mediation bypolyethylene glycol (Klebe, R. J., et al., 1983). These technologies,however, are of limited use with plants due to the presence of the plantcell wall. Other methods have been developed specifically for DNAdelivery to plant cells, such as particle bombardment (Sanford et al.,1987), silicon carbide whisker technology (Kaeppler et al., 1990), andelectroporation (D'Halluin et al., 1992). However, each of thesedelivery methods has significant limitations. For example, particlebombardment, while reported effective for transformation of some plantcells, typically relies on precipitation of DNA molecules onto thesurface of inert carrier particles prior to delivery. As a result, thisrequirement limits the usefulness of the technology for delivery ofmolecules such as proteins. In fact, there are no reports of effectivedelivery of proteins to plant cells using particle bombardment.

[0005] Silicon carbide whisker technology is reported to be much lessefficient than particle bombardment for DNA delivery to plant cells andhas been shown to be effective only in one cell type and single genotypeof corn (Frame et al., 1994). Delivery of DNA to cells viaelectroporation has been described (D'Halluin et al., 1992; Laursen etal., 1994), however, this technology is ineffective for most cell typesand there are very few reports of its successful use in planttransformation research. Furthermore, there are no known reports of itsuse to deliver proteins and other large molecules to the cells of higherplants.

[0006] Microinjection has been used to introduce proteins (Neuhaus etal., 1987) and DNA (Neuhaus, et al., 1987; U.S. Pat. No. 4,743,548) intoplant cells. The principal limitations of microinjection are that it isextremely time-consuming and possible only with cells that can beisolated and handled as single entities. For these reasonsmicroinjection has not been the method of choice for the transformationof any plant species where the goal is to produce genetically modifiedgermplasm.

[0007] Current aerosol beam technology has been reported to be capableof transforming the chloroplast genome of Chlamydomonas, a unicellular,green alga (Mets, U.S. Pat. No. 5,240,842). Chlamydomonas chloroplasttransformation can be considered a special situation since thechloroplast of Chlamydomonas is large, filling the entire cell of thetypically 10 micron size organism. However, nuclear transformation wasnot reported by Mets and the only organism reported transformed wasChlamydomonas. Furthermore, in the eight years since the technology wasfirst published, aerosol beam technology has not been reported to effectnuclear transformation of any species. Sautter et al. (1991) and U.S.Pat. No. 5,877,023, describe a technology which combines aspects of theaerosol beam and particle bombardment. Transformation with thetechnology reported by Sautter, et al., depends upon the inclusion ofgold carrier particles of 1 micron diameter. There have been no otherreports of the successful use of this technology.

[0008] As those of ordinary skill in the art recognize, it would bedesirable to introduce a range of molecules including proteins and othermacromolecules into plant and bacterial cells. This would allow, amongother possibilities, the pursuit of pioneering studies in functionalgenomics. It is clear therefore that there is a need to improve aerosolbeam technology to the point where it can be used routinely to effectnuclear transformation of important crop species such as corn andsoybean and also to introduce other large macromolecules into cells. Themethod of the present invention solves this need.

[0009] Methods of Tissue Culturinq

[0010] Cells which undergo rapid division and are totipotent aregenerally regarded as highly suitable targets for introduction of DNA asa first step in the generation of transgenic plants. Undifferentiatedcells in tissues, such as meristematic tissues and embryogenic tissuesare especially suitable. In general, cells of elite lines of crop plantsare difficult to grow in culture. Specifically, cell division afterintroduction of nucleic acid is difficult to sustain and thereforeselection of transformed cells often proves impossible.

[0011] Typically, embryogenic callus of soybean is cultured on highconcentrations of 2,4-D (Ranch et al., 1985). However, even with highconcentrations of 2,4-D in the culture medium, many cultivars do notproduce sufficient embryogenic callus for transformation experiments.Specifically, there are no reports of high frequency initiation ofcallus from immature embryos or other tissue of elite soybean lines.

[0012] The useful lifetime of a soybean variety in the marketplace isusually around three years. This does not allow time for thebackcrossing of transgenes into new and elite varieties from lines thatare not elite, since by the time this could be accomplished, newvarieties would have replaced those chosen as the recurrent parents inthe backcrossing program. Furthermore, problems with loss of yield arecommonly encountered when transgenes are introduced into elite materialfrom non-elite transformants (Minor, 1998; Oplinger, 1998). Therefore,improved culture media which are capable of supporting rapid and uniformgrowth of a range of soybean germplasm would represent a significantadvance in the art. Such an improved media are described herein.

SUMMARY OF THE INVENTION

[0013] The present invention is directed to a method for theintroduction of molecules into cells, including but not limited tobacterial and plant cells. The molecules which are introduced by themethod of the invention include, without limitation, nucleic acids,carbohydrates, plant growth regulators and peptides. The method of theinvention is further directed to the transformation of bacteria andplant cells and tissues and to the resulting transformed cells andtissues. The present invention is also directed to a method and mediumfor initiating more rapid and uniform growth of embryogenic soybeancallus, specifically the growth of elite lines of soybean.

BRIEF DESCRIPTION OF THE FIGURE

[0014]FIG. 1 is a schematic illustration of the aerosol beam apparatusof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Using the method of the present invention it is possible tointroduce molecules, including macro molecules, into cells without theneed for precipitation of the molecules onto carrier particles, andtherefore, it is not necessary to include protocols, such asprecipitation, in order to associate the molecules with carrierparticles. The present invention is especially useful for introducingpeptides into large numbers of cells allowing for studies in such areasas functional genomics. The present invention can also be used toaccomplish nuclear transformation of cells including but not limited tobacteria, and mono- and dicotyledonous plants. More particularly, elitegermplasm of crop plant species can be transformed using the presentinvention. The present invention is further directed to media andmethods for initiating rapid and uniform growth of elite lines ofsoybean, for example, transformed elite germplasm.

Definitions

[0016] The present invention employs the following definitions:

[0017] “Aerosol droplets” refer to droplets or particles, wet or dry,dispersed in a gas.

[0018] “Callus” refers to an undifferentiated mass of cells or tissue invitro.

[0019] “Carrier particles” refer to gold or tungsten or other heavymetal particles ranging in size from 0.1 micron to 4 microns which areused to introduce molecules into cells.

[0020] “Continuous Targeting” refers to the delivery of aerosol dropletsin a continuous stream toward a target.

[0021] “Elite Line” refers to a genetic line used in a product, or inthe immediate (within three years) development of a product.

[0022] “Embryogenic Callus” refers to tissue composed of large numbersof somatic embryos or embryo-like structures.

[0023] “Exogenous Gene”, “Exogenous DNA” and “Exogenous NucleotideSequence” refer to any gene, DNA or nucleic acid segment that isintroduced into a recipient cell, regardless of whether a similar genemay already be present in such a cell.

[0024] “Germplasm” refers to varieties of genetic types within aspecies.

[0025] “Genotype” refers to the genetic identity of an organism.

[0026] “Microflow Nebulizer” refers to any device that creates anaerosol when provided with a sample flow rate, with or without use of asyringe pump, of between about 1 μl/minute and about 500 μl/minute.

[0027] “Nucleotide sequence” refers to a naturally occurring ornon-naturally occurring nucleic acid, either isolated, synthesized orthe result of genetic engineering techniques.

[0028] “Phytic Acid” refers to inositolhexaphosphoric acid.

[0029] “Stage” refers to the platform on which target cells may beplaced in the method of the invention.

[0030] “Target Surface” refers to the cells comprising the uppermostlayer of cells or tissue that is first encountered by the stream ofaerosol droplets produced by the aerosol beam apparatus.

[0031] “Transformation” refers to the acquisition of new genetic codingsequences by the incorporation of an exogenous nucleotide sequence.

[0032] “Transgenic” and “Transformed” refers to organisms into whichexogenous nucleotide sequences are integrated.

[0033] The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, and immunology. See, e.g.,Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992;Glover, 1985; Anand, 1992; Guthrie and Fink, 1991; Gelvin et al., 1990;Grierson et al., 1984.

[0034] Method of Delivering Molecules to Cells

[0035] The method of the present invention offers a number of advantagesover current methods of DNA delivery. Aerosol beam technology of theinvention employs the jet expansion of an inert gas as it passes from aregion of higher gas pressure to a region of lower gas pressure througha small orifice. The expanding gas accelerates aerosol dropletscontaining the molecules to be introduced into a cell or tissue. Thesize of the droplet is of particular importance when molecules are to beintroduced into small cells or cellular organelles, for example, cellsless than about 20 microns. The importance of droplet size has beendemonstrated with particle bombardment technology where particles ofgreater than 1 micron in diameter were shown to be unsuitable forintroducing DNA into cells of 10 to 20 microns in diameter (Klein, etal., 1988(a); Klein, et al., 1988(b) due to the damage produced byparticles of such size. On the other hand, large particles have beenreported to be more likely to penetrate the cells (U.S. Pat. No.5,877,023).

[0036] Acceleration of droplets of a DNA containing solution by jetexpansion is the subject of U.S. Pat. No. 5,240,842 to Mets which isherein incorporated by reference. The only successful transformationreported by Mets is with droplets of 2 micron diameter. Droplets of thissize could be expected to destroy cells such as bacteria which aretypically no larger than 2 microns. There are no examples of successfuluse of droplets smaller than 2 microns in diameter described in thepatent to Mets. The diameter of a substantial portion of the aerosoldroplets generated by the method of the invention are believed to beless than 0.1 microns at the point of impact with the target cells. Thisis supported by the ability to routinely and efficiently transformbacterial cells of approximately 1 to 2 microns in length using themethod of the invention. Further support is found in the expectationthat droplets larger than 0.1 micron in diameter are unlikely to be ableto enter a rod-shaped or rectangular cell of 1 to 2 microns in lengthwithout destroying the cell.

[0037] Particle bombardment, as practiced using the Dupont PDS-1000,differs from aerosol beam technology in part because it relies onacceleration provided by a single burst of helium gas pressure. Viewedunder a scanning electron microscope, the particles from the Dupont gunrange in size from 0.1 microns up to 4 microns in diameter (using M5tungsten particles with an average diameter of 0.4 microns).

[0038] DNA has been introduced into bacterial cells using polyethyleneglycol (Kiebe et al., 1983), electroporation (Bonmassie et al., 1990),treatment with divalent cations (Hanahan, 1983), and particlebombardment (Smith et al., 1992). Smith et al. (1992), considerconvenience to be one of the main advantages of biolistic transformationover electroporation. Nevertheless, to achieve efficient transformationusing the biolistic process, treatment of bacterial cells with anosmoticum is required as is careful control of the relative humidity atwhich the particles were stored prior to bombardment. Bacterialtransformation using the method of the present invention is much moreefficient than biolistic-mediated transformation and requires no specialpretreatments.

[0039] Based on successful and efficient transformation of bacteriausing the method of the present invention, it is believed that asubstantial number of the aerosol droplets produced are less than 0.1micron in diameter at the point of impact with the target cells. DNAcarried in aerosol droplets of this small size penetrates cells onlybecause of the speeds attained by the aerosol droplets. Speeds achievedby the aerosol beam method of the invention are supersonic and can reach2000 meters/second. By contrast, top speed achieved by the particle gunis 200 meters/second.

[0040] In part, because aerosol droplets generated by the presentinvention are so much smaller than the particles produced by the priorart, the present invention is superior in delivering molecules to smallcells, for example cells less than 20 microns in diameter. Many animal,plant and microbial cells are in this size range. Entry of micron-sizeparticles into cells of this size can be expected to cause significantdamage. The very small aerosol droplets produced by the method of thepresent invention are also advantageous for chloroplast transformation,particularly in monocots and graminaceous embryogenic cell culturescontain proplastids (the target for chloroplast transformation) that aretypically less than 1 micron in diameter (Bilang and Potrykus, 1998).

[0041] Another advantage of the present invention is that it permitsbetter quantification of DNA delivery to cells than is possible withparticle gun technology. This is because no precipitation or coating ofDNA onto carrier particles is required with the method of the invention,procedures which introduce variability into the DNA delivery process.

[0042] As a further embodiment of the present invention, molecules otherthan DNA can be readily introduced into cells, either alone or incombination with DNA. Inclusion of molecules other than DNA introducedat the same time as DNA could improve integration and increase thenumber of selectable transformants. By contrast, effective delivery ofmolecules can be achieved with particle bombardment only after firstcoating the particles with the substance to be delivered. In those caseswhere it is not possible or desirable to use coated particles, theaerosol beam may be the most effective way to deliver chemicals directlyto the interior of cells on a large scale.

[0043] An additional advantage of the present invention is that DNA isdelivered as a stream of aerosol droplets emerging through a smallorifice (continuous targeting). This targeting can continue for as longas the target tissue can survive being held in a vacuum. In the courseof delivery the droplet stream can then be precisely targeted. Thisfurther distinguishes aerosol beam technology from particle bombardmentas currently practiced where all DNA-coated particles are delivered tocells in a single blast resulting in a shot pattern covering an area ofseveral centimeters in diameter. A hybrid of the aerosol beam andparticle bombardment methods (Sautter, et al.; U.S. Pat. No. 5,877,023)is reported to deliver a solution of DNA and 1 micron diameter goldcarrier particles in interrupted blasts, thus continuous targeting oftissue is not possible with this method. Continuous targeting alsoallows for the identification of an area of tissue (such as the apex ofa shoot meristem) and its positioning under the beam to ensure focuseddelivery of aerosol droplets to the tissue. Furthermore, repeated blastswhich are delivered with particle bombardment and the method of Sautter,et al., can be expected to result in severe and extensive tissuedestruction. The ability to continuously target cells or tissues makesthe aerosol beam clearly superior to other direct DNA delivery methodsincluding particle bombardment.

[0044] The aerosol beam of the present invention differs from U.S. Pat.No. 5,240,842 in numerous respects. First, Mets does not include anypositive pressure entrainment airflow to guide or focus the aerosolbeam. Second, Mets includes a vent in the compressed gas path to allowrelease of excess aerosol. This results in wasted compressed gas andaerosol material. The vent is required in Mets because of the high flowrates used. Third, the nebulizer used by Mets is a type used ininhalation therapy and is described as of the Lovelace design. Thisnebulizer is a single use disposable unit that generates aerosoldroplets with median mass diameters in the range of 2 microns.Commercially available nebulizers such as HEN or MCN100 were used inexample. However, any microflow nebulizer, as defined herein, can beused in the practice of the present invention. Unexpectedly, use ofmicroflow nebulizers cacilitated iinsertion of molecules into cells.Fourth, the preferred embodiment of the present invention includes asyringe pump which regulates the flow rate of the sample to thenebulizer. Fifth, droplets of the size described by Mets would be toolarge to deliver DNA or any other molecule to most bacteria or plantcells and yet allow survival of these cells.

[0045] Transformation of Plant Cells and Tissues

[0046] Introduction of DNA and other molecules such as proteins intoplant cells by the method of the present invention is exemplified bytransformation of corn (monocotyledonous plants) and soybean(dicotyledonous plants). Briefly, the transgenic plants of thisinvention may be produced by (I) culturing a source of cells, (II)optionally, pretreating cells to yield tissue with increased capacityfor uptake and integration by aerosol beam technology, (III)transforming said tissue with an exogenous nucleotide sequence by theaerosol beam method of the invention, (IV) optionally, identifying orselecting for transformed tissue, (V) regenerating transgenic plantsfrom the transformed cells or tissue, and (VI) optionally, producingprogeny of said transgenic plants.

[0047] Method of Culturing Cells

[0048] Corn Cell Cultures.

[0049] The corn cells which may be used as starting materials in thepresent transformation process include elite inbred lines of corn. Forexample, embryogenic callus and immature embryos of Stine Inbred 963were used for both transient expression experiments and the productionof stably transformed callus, embryos and plants. Other cells may beused, including those derived from meristems. These meristems are found,for example, in juvenile leaves, immature tassels, immature and matureembryos and coleoptilar nodes. While the method of the present inventioncan be applied to any corn cells from which fertile plants can berecovered, cell cultures derived from immature embryos or embryogeniccallus have been utilized herein for purposes of example.

[0050] Methods of preparing and maintaining corn cells are well known inthe art (Duncan et al., 1985). Typically, cultures are prepared fromimmature maize embryos which have been removed from the kernels of anear when the embryos are about 1-2 mm. in length. The scutellum ofimmature embryos can be stimulated to give rise to embryogenic callusfrom which plants can be regenerated. Embryogenic callus can also beobtained from the developing reproductive organs of a corn plant.Exemplary methods for isolating immature embryos from corn are describedby Green and Phillips (1976).

[0051] In a preferred embodiment of the invention, the embryos wereplaced on culture medium, for example, DN62AG (Table 1), under asepticconditions. This culture medium, DN62AG, has been described in U.S. Ser.No. 09/203,679, filed December, 1998, incorporated herein by reference.It has been discovered that immature embryos incubated for approximately2 to 3 days on DN62AG medium, after dissection and prior to beaming,show improved survival. This, in turn, improves the efficiency withwhich transformants can be recovered. The embryogenic callus culturesare routinely maintained on stock culture medium, preferably on DN62(Table 1) for ten-day periods between transfers.

[0052] Soybean Cell Cultures.

[0053] Soybean cells which can be used as starting materials in themethod of the invention include cell cultures and explants containingmeristematic tissue from which plants can be regenerated. Exemplary ofcells which are appropriate are embryogenic callus of Stine 13404-TT(Examples 10).

[0054] Conventional methods of preparing and maintaining embryogenicsoybean cultures are described by Ranch et al. (1985). In one embodimentof the invention, the medium of Ranch et al (1985), can be modified bythe inclusion of one or more of four constituents (Example 8). Immaturecotyledons about 1 mm in length were used as the initial explants andwere placed on culture media under sterile conditions.

[0055] In another embodiment of the present invention, a novel culturemedia may be used to stimulate high frequency production of embryogenicsoybean callus. Improvement varied with the genotype being cultured. Thelength of time required for a culture passage was unexpectedly reducedto two weeks with the use of this novel medium as compared to four weekstypical with other media. The inclusion of one or more of four mediaconstituents, coconut water, myoinositol, phytic acid and inorganicphosphate concentration, enhanced embryogenic callus production andallowed significant improvements to be made to transgenic cloneproduction in terms of number of clones recovered, embryo morphology,and reduction in the time needed to identify the clones and regenerateplants from them. The medium of Ranch et al., 1985 (referred to hereinas B1-30) was used as the basal medium. An example of the medium of thepresent invention is B1-30 3Co5My0.25PA0.5K (footnote, Table 5).Although this medium is a preferred embodiment of growth medium, otherconventional media may be utilized in the practice of the invention.

[0056] Pretreatment of Cells

[0057] In another embodiment of the present invention, pretreatment ofthe cells may be carried out in order to increase nucleic acid deliveryusing the method of the invention. For example, corn cells can beosmotically stressed to improve DNA delivery while preserving cellviability. Possible methods of osmotic stress include those conventionalmethods known in the art, for example, Russell et al., 1992. As apreferred embodiment of the invention, a novel medium designed toprovide osmotic stress referred to as DN62OSM was used (Example 2, Table1). The duration of exposure to osmotic stress may range from about 45minutes up to about 24 hours on this medium, with a preferred durationof about 45 minutes to about one hour. In the case of soybean cells anosmotic pretreatment was not employed.

[0058] Introduction of Nucleic Acid into Cells

[0059] One embodiment of the present invention is directed to theintroduction of a nucleotide sequence into plant cells via aerosol beamtechnology. Once a desired nucleotide sequence has been synthesized orcloned, and engineered, it is necessary to integrate it into the genomeof a plant of interest so that it is stably inherited by progeny of thetransformed plant. Following introduction of foreign sequence intotarget cells and subsequent cell division, selection is applied toidentify those cells in which integration and expression of the sequenceis occurring. Stable transformation involves the integration offunctional genetic sequences into the genome so that the integratedsequences are passed on to and are present in the transformed plants.Any procedure which could enable the stable integration of nucleic acidswould greatly improve transformation protocols. In contrast, transienttransformation results in eventual loss of the sequence and, therefore,transient methods are of little use in generating transgenic plants,although they may be of use in the optimization of conditions for stabletransformation and in evaluation of gene expression.

[0060] An embodiment of the aerosol beam apparatus of the presentinvention is shown in FIG. 1. The apparatus includes a pressurized gassupply 10, a vacuum chamber 13, and an entrainment housing 11.

[0061] The pressurized gas supply 10 may contain a pressurizedpropellant gas such as, for example, helium. The pressurized gas supply10 is connected to a microflow nebulizer conduit 7 and is also connectedto an entrainment tube 5.

[0062] The entrainment housing 11 has an interior, and located withinthe interior is all or a portion of a microflow nebulizer 8. Theentrainment housing 11 interior communicates with the pressurized gassupply 10 via the entrainment tube 5, which may include a pressureregulator 20. The microflow nebulizer 8 communicates with thepressurized gas supply 10 via the microflow nebulizer conduit 7, whichmay include a filter 16 and a pressure regulator 19. A single regulatormay be employed if the entrainment gas pressure can be the same as thenebulizer gas pressure. The entrainment housing 11 may also include atemperature controller 17 which controls the temperature in theentrainment housing 11 to a range of about 32 degrees to about 80degrees Centigrade. A pressure gauge 6 is connected to the entrainmenthousing 11 and may be used to control the pressure regulator 20 to setthe entrainment air pressure and flow. The entrainment housing 11 mayinclude a nucleospot 9. The nucleospot 9 may be used to reduceelectrostatic charges created by the moving gas.

[0063] The microflow nebulizer 8 is an aerosol nebulizer that is fedboth a sample material and a pressurized gas. The sample material may befed to the microflow nebulizer 8 by a pump or other suitable means. Inthe microflow nebulizer 8, the pressurized gas forces the samplematerial through a small orifice in the nebulizer, where the pressurizedgas converts the sample material into aerosol droplets. The aerosoldroplets are then carried by the resulting beam of gas to the nozzle,through which the aerosol droplets greatly accelerated.

[0064] A sample material supply conduit 4 is connected to the microflownebulizer 8. The sample material supply conduit 4 may be furtherconnected to a pump 2 and filter 3. In a preferred embodiment, the pump2 is a syringe pump and includes a plastic syringe 1 holding a quantityof an sample material.

[0065] In addition to the aerosol beam, pressurized helium flows intothe entrainment housing 11 through the entrainment tube 5. Thisentrainment gas flow also has a velocity and moves substantiallyparallel to the aerosol spray, and serves to entrain the aerosol sprayand focus it on the way to the target.

[0066] The vacuum chamber 13 includes a nozzle 12, a vacuum pump 15, anda door 22. The vacuum chamber 13 may additionally include a stage 14, avacuum gauge 21, and a stage switch 18. The nozzle 12 further guides theaerosol beam as it approaches the stage 14 (on which the target to beinjected rests). The nozzle 12 includes an orifice, with the orificesize ranging from about 200 microns to about 500 microns. The orificesize is preferably 300-330 microns. The combination of the pressurizedgas in the entrainment housing 11 and the partial vacuum in the vacuumchamber 13 accelerates the aerosol droplets that impact the targetcells.

[0067] The stage 14 may be a movable stage wherein the target may bemoved relative to the incoming aerosol spray so that a controlled areamay be impacted with the sample material. The stage switch 18 may beused to control movement of the stage 14. The vacuum gauge 21 may beused to monitor and control the vacuum level in the vacuum chamber 13.

[0068] The aerosol can be produced by various microflow nebulizers knownin the art, such as the HEN from J.E. Meinhard Associates, Inc., or theMCN100 style M-4 nebulizer from Cetac Technologies, Inc., although othermicroflow nebulizers may also be used. The preferred nebulizer describedin U.S. Pat. No. 5,240,842 was one typically used in inhalation therapyand was described as being of the Lovelace design which is a single-usedisposable unit that generates aerosol droplets with median massdiameters in the range of 2 microns.

[0069] The nebulizing gas can be selected from those inert gases knownin the art, preferably high purity compressed helium. The gas isregulated and filtered. The entrainment gas can be high puritycompressed helium, filtered or not. The entrainment tube or housing maycontain a nucleospot to reduce electrostatic charges and can bemaintained at a temperature of between about 32 and 80° C. bytemperature controllers. The sample flow rate may be set at from about 1to about 1200 μl/min.

[0070] The method of the present invention differs from the severalembodiments described in Mets (U.S. Pat. No. 5,240,842) wherein a commonfeature was the presence of a vent to allow release of excess aerosolwhich was the result of the high flow rates that were used. It has beenunexpectedly discovered that high efficiency microflow nebulizers, suchas the HEN and MCN 100, provide adequate aerosol droplet production whenusing very low (1 to 350 μl/min) flow rates. Venting of excess aerosolis therefore not necessary with the method of the invention.Furthermore, the aerosol droplets produced by the microflow nebulizersin the method of the invention are much smaller at the point of impactwith the target than the 2 micron diameters preferred by Mets. Smalldroplet size can be maintained in the method of the invention by the useof helium as the nebulizing gas although other means known in the artmay be used.

[0071] Improved efficiency of transformation is possible using themethod of the invention which employs the routine production of smallaerosol droplets traveling at supersonic speed. The correlation betweenthe reduced droplet size and improved efficiency was unexpected. It waspreviously reported in U.S. Pat. No. 5,877,023, in regard to particlesize, that large particles are more able to penetrate the cells thoughuse of small particles is more favorable for the survival of cells (Col.7, L. 14-18). The method of the invention also improves upon existingtechnology by enabling the routine transformation of bacteria.

[0072] The chamber vacuum can be maintained at from about 26 to about 30in. Hg throughout a given run with use of a vacuum pump. Water may beplaced in the vacuum chamber to prevent loss of moisture from the targettissue. A syringe needle cut off just proximal to the plastic holder canbe used for the nozzle. Nozzle diameters of from about 200 to about 500microns can be used. The target tissue can be placed in the center of anagar plate below the nozzle tip. The stage movement can be adjusted toachieve the desired result.

[0073] Briefly, treatment of target tissue with the aerosol beamapparatus may be performed as follows: (1) place tissue on targetsurface, on the stage; (2) start the vacuum pump; (3) set the nebulizinggas pressure; (4) set the entrainment gas pressure; (5) start thesyringe pump; (6) start the movement of the stage and let it run whilethe aerosol particles suspended in the inert gas impact the targettissue. Delivery can continue for as long as the target tissue cansurvive being held in a vacuum.

[0074] In order to successfully produce stably transformed plants byaerosol beam technology, four requirements must be met: (1) the targetcells must remain viable; (2) the target cells must be able to take upthe recombinant nucleic acid at high enough frequencies to insure thestable transformation of a useful number of cells; (3) once transformed,the recipient cells must be able to maintain cell division andregenerative capacity throughout the selection process in order toconfirm and identify stably transformed cells; and (4) the transformedregenerated plants must be able to express the recombinant nucleic acid.

[0075] Utilizing the method of the present invention, accurate andextensive tissue targeting can be achieved with any explant, includingimmature embryo, immature tassel, section of leaf or root, anther,pollen and meristem cells of corn, and meristem and somatic embryo cellsof soybean. For example, in the case of corn, pollen, as well as itsprecursor cells, microspores, may be capable of functioning as recipientcells for nucleic acid delivery, or as vectors to carry foreign nucleicacid for incorporation during fertilization. The continuous targetingwhich is possible with the method of the invention enables flexibilityand accuracy in delivering nucleic acid to target cells. Individualimmature corn embryos can be targeted so that only particular regions ofthe scutellum are subjected to the aerosol beam or, alternatively, it ispossible to deliver nucleic acid to the entire surface area of thescutellum with the aerosol beam.

[0076] Examples of genes useful for expression in transformed plantcells are known in the art. More particularly, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Suchgenes include, but are not limited to, those described herein.

[0077] Genes that Confer Resistance or Tolerance to Pests or Disease

[0078] (A) Plant disease resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance (R) gene in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant variety can betransformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. Examples of such genes include,the tomato Cf-9 gene for resistance to Cladosporium fulvum (Jones et al.(1994)), the tomato Pto gene, which encodes a protein kinase, forresistance to Pseudomonas syringae pv. tomato (Martin et al. (1993)),and the Arabidopsis RSSP2 gene for resistance to Pseudomonas syringae(Mindrinos et al. (1994)).

[0079] (B). A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon, such as, a nucleotide sequence ofa Bt δ-endotoxin gene (Geiser et al. (1986)). Moreover, DNA moleculesencoding δ-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), under ATCC accession numbers. 40098, 67136,31995 and 31998.

[0080] (C) A lectin, such as nucleotide sequences of several Cliviaminiata mannose-binding lectin genes (Van Damme et al. (1994)).

[0081] (D) A vitamin binding protein, such as avidin and avidin homologswhich are useful as larvicides against insect pests. See U.S. Pat. No.5,659,026.

[0082] (E) An enzyme inhibitor, e.g., a protease inhibitor or an amylaseinhibitor. Examples of such genes include a rice cysteine proteinaseinhibitor (Abe et al. (1987)), a tobacco proteinase inhibitor I (Huub etal. (1993)), and an α-amylase inhibitor (Sumitani et al. (1993)).

[0083] (F) An insect-specific peptide or neuropeptide which, uponexpression, disrupts the physiology of the affected pest. Examples ofsuch genes include, an insect diuretic hormone receptor (Regan (1994),an allostatin identified in Diploptera puntata (Pratt (1989)),insect-specific, paralytic neurotoxins (U.S. Pat. No. 5,266,361).

[0084] (G) An insect-specific venom produced in nature by a snake, awasp, etc., such as, a scorpion insectotoxic peptide (Pang (1992)).

[0085] (H) An enzyme responsible for a hyperaccumulation of amonoterpene, a sesquiterpene, a steroid, hydroxamic acid, aphenylpropanoid derivative or another non-protein molecule withinsecticidal activity.

[0086] (I) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, anuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. Examples ofsuch genes include, a callas gene (PCT published applicationWO93/02197), chitinase-encoding sequences (which can be obtained, forexample, from the ATCC under accession numbers 3999637 and 67152),tobacco hookworm chitinase (Kramer et al. (1993)) and parsley ubi4-2polyubiquitin gene (Kawalleck et al. (1993)).

[0087] (J) A molecule that stimulates signal transduction. Examples ofsuch molecules include, nucleotide sequences for mung bean calmodulincDNA clones (Botella et al. (1994)), a nucleotide sequence of a maizecalmodulin cDNA clone (Griess et al. (1994)).

[0088] (K) A hydrophobic moment peptide. See U.S. Pat. Nos. 5,659,026and 5,607,914, the latter teaches synthetic antimicrobial peptides thatconfer disease resistance.

[0089] (L) A membrane permease, a channel former or a channel blocker,such as, a cecropin-β lytic peptide analog (Jaynes et al. (1993)) whichrenders transgenic tobacco plants resistant to Pseudomonas solanacearum.

[0090] (M) A viral protein or a complex polypeptide derived therefrom.For example, the accumulation of viral coat proteins in transformedplant cells imparts resistance to viral infection and/or diseasedevelopment effected by the virus from which the coat protein gene isderived, as well as by related viruses. Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus. See, for example, Beachy et al. (1990).

[0091] (N) An insect-specific antibody or an immunotoxin derivedtherefrom. Thus, an antibody targeted to a critical metabolic functionin the insect gut would inactivate an affected enzyme, killing theinsect. For example, Taylor et al. (1994) shows enzymatic inactivationin transgenic tobacco via production of single-chain antibody fragments.

[0092] (O) A virus-specific antibody. See, for example, Tavladoraki etal. (1993), which shows that transgenic plants expressing recombinantantibody genes are protected from virus attack.

[0093] (P) A developmental-arrestive protein produced in nature by apathogen or a parasite. Thus, fungal endo α-1,4-D polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-α-1,4-D-galacturonase (Lamb et al.(1992)). The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.(1992).

[0094] (Q) A developmental-arrestive protein produced in nature by aplant, such as the barley ribosome-inactivating gene, have increasedresistance to fungal disease (Longemann et al. (1992)).

[0095] Genes that Confer Resistance or Tolerance to a Herbicide

[0096] (A) A herbicide that inhibits the growing point or meristem, suchas an imidazalinone or a sulfonylurea. Exemplary genes in this categorycode for mutant ALS (Lee et al. (1988)) and AHAS enzyme (Miki et al.(1990)).

[0097] (B) Glyphosate (resistance imparted by mutant EPSP synthase andaroA genes) and other phosphono compounds such as glufosinate (PAT andbar genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor encoding genes). See, for example, U.S. Pat. No.4,940,835, which discloses the nucleotide sequence of a form of EPSPsynthase which can confer glyphosate resistance. A Nucleic acid moleculeencoding a mutant aroA gene can be obtained under ATCC accession number39256, and the nucleotide sequence of the mutant gene is disclosed inU.S. Pat. No. 4,769,061. European patent application No. 0 333 033 andU.S. Pat. No. 4,975,374 disclose nucleotide sequences of glutaminesynthase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a phosphinothricinacetyltransferase gene is provided in European application No. 0 242246. De Greef et al. (1989) describes the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al. (1992).

[0098] Genes that Confer Resistance or Tolerance to EnvironmentalStresses

[0099] (A) Cold, freezing or frost. This includes genes that code forproteins that protect from freezing and for enzymes that synthesizecryoprotective solutes. Examples of such genes are Arabidopsis COR15a(Artus et al. (1996)) and spinach CAP160 (Kaye et al. (1998)). Also inthis category are regulatory genes that control the activity of othercold tolerance genes (Tomashow and Stockinger (1998)).

[0100] (B) Drought or water stress. Kasuga et al. (1999) report howstress inducible expression of DREB1A in trangenic plants increasestheir tolerance of drought stress. Pilin-Smits et al. (1998) report thatexpression of baterial genes for synthesis of trehalose producestolerance of water stress in transgenic tobacco.

[0101] (C) Salinity or salt stress. Genes that code for proteins thatminimize uptake of sodium in the presence of high salt, or cause theplant to sequester sodium in vacuoles, can enable plants to toleratehigher levels of salt in the soil. The wheat HKT1 potassium transporter,described by Rubio et al. (1999), is an example of the former. Apse etal. (1999) describe how an Arabidopsis Na⁺/H⁺ antiporter can act in thelatter manner.

[0102] (D) Metals. Protection from the toxic effects of metals such asaluminum and cadmium can be accomplished by transgenic expression ofgenes that prevent uptake of the metal, or that code for chelatingagents that bind the metal ions to prevent them from having a toxiceffect. Examples of such genes are Arabidopsis ALR104 and ALR108 (Larsenet al. (1998)) and genes for the enzymes involved in phytochelatinsynthesis (Schafer et al. (1998)).

[0103] Genes that Confer or Contribute to a Value-Added Trait

[0104] (A) Modified fatty acid metabolism, for example, by transformingmaize or Brassica with an antisense gene or stearoyl-ACP desaturase toincrease stearic acid content of the plant (Knultzon et al. (1992)).

[0105] (B) Decreased Phytate Content

[0106] (1) Introduction of a phytase-encoding gene would enhancebreakdown of phytate, adding more free phosphate to the transformedplant, such as the Aspergillus niger phytase gene (Van Hartingsveldt etal. (1993)).

[0107] (2) A gene could be introduced that reduces phytate content. Inmaize, for example, this could be accomplished by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid (Raboy etal. (1990)).

[0108] (C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. Examples of such enzymes include,Streptococcus mucus fructosyltransferase gene (Shiroza et al. (1988)),Bacillus subtilis levansucrase gene (Steinmetz et al. (1985)), Bacilluslicheniformis α-amylase (Pen et al. (1992)), tomato invertase genes(Elliot et al. (1993)), barley amylase gene (Søgaard et al. (1993)), andmaize endosperm starch branching enzyme II (Fisher et al. (1993)).

[0109] (D) Modified lignin content. The amount or composition of lignincan be altered by increasing or decreasing expression of thebiosynthetic enzymes for phenylpropanoid lignin precursors, such ascinnamyl alcohol dehydrogenase (CAD), 4-coumarate:CoA ligase (4CL), andO-methyl transferase (OMT). These and other genes involved in formationof lignin are described in Bloksberg et al. (1998).

[0110] As those of ordinary skill in the art will recognize, this isonly a partial list of possible genes that can be used with thetransformation method of the present invention. Synthesis of genessuitably employed in the present invention can be effected by means ofmutually priming long oligonucleotides. See, for example, Ausubel et al.(1990) and Wosnick et al. (1987). Moreover, current techniques whichemploy the polymerase chain reaction permit the synthesis of genes aslarge as 6 kilobases in length or longer. See Adang et al. (1993) andBambot et al. (1993). In addition, genes can readily be synthesized byconventional automated techniques.

[0111] As those of ordinary skill in the art will also recognize,regulatory sequences including promoters, terminators and the like willalso be required, and these are generally known in the art (Zhao et al.(1998)). Plant expression cassettes preferably comprise a structuralgene to which is attached regulatory DNA regions that permit expressionof the gene in plant cells. The regulatory regions consist at a minimumof a promoter capable of directing expression of a gene in a plant cell.The promoter is positioned upstream or at the 5′ end of the gene to beexpressed. A terminator is also provided as a regulatory region in theplant expression cassette and is capable of providing polyadenylationand transcription terminator functions in plant cells. The terminator isattached downstream or at the 3′ end of the gene to be expressed. Markergenes, included in the vector, are useful for assessing transformationfrequencies in this invention.

[0112] The nucleic acid to be delivered to cells may contain selectablemarker sequences under control of appropriate recognizable promoters foruse in selecting transformed cells. Numerous selectable marker genes areavailable for use in plant transformation including, but not limited to,neomycin phosphotransferase II, hygromycin phosphotransferase, EPSPsynthase and dihydropteroate synthase. See, Miki et al. (1993). Othermarkers and promoters are well known in the art. The vectors may alsocontain selectable marker sequences under control of appropriaterecognizable promoters for use in selecting transformed cells. Suitablemarkers and promoters are well known in the art.

[0113] The recombinant nucleic acid used for transformation herein maybe circular or linear, double-stranded or single-stranded. Generally,the nucleic acid is in the form of a recombinant plasmid and containscoding regions of beneficial heterologous nucleic acid with flankingregulatory sequences which promote the expression of the nucleic acid inthe genome of the transformed plant. For example, the recombinantnucleic acid may itself comprise or consist of a promoter that is activein other genotypes, or may utilize a promoter already present in thegenotype that is the transformation target.

[0114] The compositions of, and methods for, constructing a nucleic acidsequence which can transform certain plants are well known to thoseskilled in the art, and the same compositions and methods ofconstruction may be utilized to produce the recombinant nucleic aciduseful herein (J. Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press (2d), 1989). The specificcomposition of the nucleotide sequence is not central to the presentinvention and the invention is not dependent upon the composition of thespecific transforming nucleotide sequence which is used.

[0115] Restriction enzymes can be introduced, using the method of theinvention, along with linear nucleic acid having compatible cohesiveends to increase the frequency of transformants. The introduction ofrestriction enzymes andn via electroporation has been reported for yeastand Dictyostelium (Schiestl and Petes, 1991; Kuspa and Loomis, 1992).However similar results have not previously been reported with planttransformation.

[0116] Selection of Transformed Cells

[0117] After beaming, the tissue may be retained on the osmoticum orshortly thereafter transferred to a recovery medium. Following recovery,it is desirable to identify and select those cells which contain theexogenous nucleic acid. There are two general approaches which have beenfound useful for accomplishing this. First, the transformed cells can bescreened for the presence of the recombinant nucleic acid by variousstandard methods which could include assays for the expression ofreporter genes, use of probes for or amplification of the desirednucleotide sequence and assessment of phenotypic effects of therecombinant nucleic acid, if any. Alternatively and preferably, when aselectable marker or reporter gene has been transmitted along with or ispart of the recombinant nucleic acid, those cells which have beentransformed can be identified by detecting expression of the selectablemarker or reporter genes. For example, transient expression may beevaluated by use of the GUS expression cassette containing the GUS gene,which encodes an enzyme for which various chromogenic substrates areknown. Stable transformation may be evaluated by use of the barexpression cassette. Various other selection schemes for identificationof stably transformed tissue may be used, including selection onbialaphos or use of the GFP gene in combination with the Streptomycesbar gene, allowing for visual selection of fluorescing transformedtissues.

[0118] Regeneration of Transformed Cells

[0119] Conventional regeneration methods, well known in the art, may beused for corn species. (Duncan et al., 1985; U.S. Pat. Nos. 5,484,956;5,489,520; 5,177,010; 5,641,664; and 5,350,689, all incorporated hereinby reference.)

[0120] Soybean regeneration may be achieved either by the methods ofRanch et al., 1985, or preferably by the methods disclosed herein(Example 9).

EXAMPLES

[0121] The present invention is further detailed in the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the technique specifically described below are utilized.

Example 1 Delivery of Molecules into Cells and Tissues by the Method ofthe Invention

[0122] Aerosol droplets containing nucleic acids and/or proteins wereintroduced into target tissue such as cells using the aerosol beamapparatus of the invention (FIG. 1). The aerosol was produced by amicroflow nebulizer such as the HEN from J. E. Meinhard Associates Inc.,or the MCN100 style M4 nebulizer from Cetac Technologies Inc. (Liu andMontaser, 1994; Tan, et al., 1992). The nebulizing gas was high puritycompressed-helium which was regulated with an ACCU-TROL gasregulator-876X model RS-7-4 and filtered through an Arrow F300-02 ITfilter. When HEN and the MCN100 microflow nebulizers were used, thenebulizing pressure was preferably 20-30 psi but worked within the rangefrom about 10 psi to about 40 psi. The entrainment gas filled theentrainment tube and entrained the aerosol droplets in a straight line.Unfiltered, high purity compressed helium was used as the entrainmentgas and was regulated by an Arrow R262 regulator to produce slightpositive pressure as measured by a Gilmont model 65 mm gauge. Theentrainment housing contained a nucleospot to reduce electrostaticcharges and was maintained at a temperature of about

[0123] 42° C. to about 55° C., and most preferably about 55° C. Thisreduced coalescing of the aerosol droplets and was controlled by twoOmega CN9000 series temperature controllers. The sample flow rate to thenebulizer was controlled by a Harvard 11 infusion only syringe pump. Theflow rate was 1 to 1200 μl/min using a sterile Becton Dickinson 1 ccplastic syringe with a 0.2 micron filter attached. The sample contained10 mM Tris buffer (pH 7.0) or a carbohydrate molecule (for example 1 g/lsucrose) and the molecules to be delivered.

[0124] As discussed previously, venting of excess aerosol which isreported in U.S. Pat. No. 5,240,842 is not necessary with the method ofthe invention. Unexpectedly, venting drastically reduced effectivenessof the method of the invention. The target tissue was placed onsolidified agar medium in a petri dish on the stage, about 3 cm belowthe nozzle tip. The stage was mounted on a XY-4040 precision grade table(New England Affiliated Technologies) which was controlled by LabVIEW5.0 software (National Instruments). The stage moved in a straight lineat the rate of 20-80 mm/sec with about 0.3 mm to 1.0 mm distance betweenpasses. Typically a run of 1.0-3.0 minutes was performed which coveredabout a 2.0 to 4.0 cm diameter area of target tissue. The chamber vacuumwas maintained at around 26 to about 30 inches Hg throughout a given runby a Welch 1405 DuoSeal vacuum pump. The vacuum created a pressuredifferential that was mediated through the nozzle. When the aerosoldroplets produced by the nebulizer in the chamber of comparably highpressure passed through the nozzle, they greatly accelerated into thevacuum of the lower chamber. A small beaker of water was placed in thevacuum chamber to prevent loss of moisture from the target tissue. Thenozzle was preferably a luer-lock 24 gauge (305 um inside diameter)Becton Dickinson syringe needle cut off just proximal to the plasticholder. However, syringe diameters of about 200 to about 500 microns aswell as nozzles known in the art, other than syringes, can be used.

[0125] Parameters will vary for particular plant tissues. Tissue whichcan withstand the damage caused by the beam may produce moretransformants when subjected to a more intense barrage of aerosoldroplets (produced by using a wider orifice, by slowing stage speed, orby decreasing the distance between passes, for example).

[0126] Briefly, the treatment of target tissue with the aerosol beamapparatus was performed as follows: 1) place petri dish with tissue onthe stage and close vacuum chamber; 2) start the vacuum pump; 3) startthe syringe pump; 4) set the nebulizing gas pressure; 5) set theentrainment gas pressure, and by this time the correct vacuum in thechamber is reached; and 6) start the movement of the stage and let thesystem run for the time needed to complete the run. After the run iscompleted, shut down the stage, vacuum, syringe pump, nebulizing gas,entrainment gas, and remove target tissue from the chamber.

Example 2 Introduction of Nucleic Acid into Corn Callus and Detection ofTransient Expression

[0127] Embryogenic corn callus of Stine inbred 963 was transferred fromstock culture maintenance medium, DN62 (Table 1) to a medium formulatedto provide osmotic stress to the tissue. A preferred embodiment of theinvention, DN62OSM medium was used (Table 1). Preferably the embryogeniccallus was transferred two to three days after transfer to freshmaintenance medium, a time when the cells are dividing rapidly (formaintenance, cultures are routinely transferred every ten days). Afterat least 45 minutes (preferably an hour) and up to 24 hours on thismedium, tissue was collected and oriented in the center of the targetsurface prior to beaming. After beaming, the tissue was allowed toincubate on DN62OSM medium for one day.

[0128] The plasmid used in transient expression experiments waspreferably pBARGUS which was obtained from the Plant Gene ExpressionCenter, Albany, Calif., although other plasmids known in the art can beused. Plasmid BARGUS contains a beta-glucuronidase (GUS) expressioncassette consisting of a GUS gene driven by the corn adh1 promoter andadh1 intron1 terminated with a nos terminator, and a bar expressioncassette consisting of a bar gene driven by a CaMV 35S promoter and anadh1 intron terminated with a nos terminator. The GUS expressioncassette is used to detect transient expression while the bar expressioncassette confers tolerance to the herbicide bialaphos. Thus, if desired,selection of stably transformed clones could be achieved. Anotherplasmid used in transient assays was p350096, which has the CaMV 35Spromoter, alcohol dehydrogenase intron six (IV6) driving the GUS genewhich is terminated with the nopaline synthase (nos) 3′ end.Approximately two micrograms of supercoiled plasmid DNA was added to 1.0ml of the buffered solution, however, higher and lower concentrations ofDNA can be used. For example, DNA concentrations as high as about 20μg/ml of DNA and as low as about 0.1 μg/ml to about 1.0 μg/ml wereeffective, although delivery was less effective when using 0.1 μg/ml. Interms of number of DNA molecules, there are approximately 99 billionmolecules in 1 ug of pBARGUS DNA. A DNA concentration of 0.1 ug/mlproduced less transient expression than did 2.0 ug/ml while a DNAconcentration of 1.0 ug/ml produced similar transient expression to 2.0ug. Higher concentrations than 2.0 ug produced marginally more intensetransient expression. Supercoiled or linear DNA could be used intransient expression experiments, however, in this example supercoiledwas preferred because of the ease of preparation. Plasmid DNA wasisolated using Qiagen midi or maxi preps as described by themanufacturer.

[0129] The aerosol beam procedure used to transform corn callus wasessentially as previously described (Example 1). Preferred parametersincluded an entrainment tube or housing maintained at about 55° C. withthe sample solution flow rate set at about 0.5 ml/hour to about 1.0ml/hour (8 μl/min. to about 17 μl/min.) and the vacuum chamber pressuremaintained at about 29 inches Hg. Treatment of target tissue with theaerosol beam typically continued for about one to about three minutes,however, beaming can continue for as long as the target tissue cansurvive being held in a vacuum which, in the case of immature cornembryos and embryogenic callus, is at least 3 minutes.

[0130] Sterile technique was used routinely to prevent contamination oftarget tissue. A seventy percent ethanol solution was sprayed on theinside of the vacuum chamber and entrainment tube prior to the start ofan experiment and the tubing attached to the nebulizer and the microflownebulizer itself were rinsed out with 70% ethanol which was followed bya wash with sterile water before adding the sample solution. Sterilewater was replaced with the sample solution before treating the targettissue with the aerosol beam apparatus.

[0131] Approximately one day after treating the corn callus with theaerosol beam apparatus, transient expression was evaluated byhistochemical analysis. Embryogenic callus was incubated in the presenceof the substrate X-gluc (Gold Biotechnology, Inc.) at a concentration of0.5 mg/ml in 0.1 M sodium phosphate buffer pH 7.0 and 0.1% Triton-x-100at 37° C. After 1-4 hours blue spots appeared indicating GUS expressionwhich verifies that the GUS expression cassette was introduced into thecell with the aerosol beam apparatus. Transient expression results wereused to compare the efficiency of DNA delivery, using equivalent amountsof DNA, to embryogenic callus using either the method of the inventionor a DuPont PDS 1000 particle delivery device. Delivery using the methodof the invention was readily observed to be more efficient in terms ofthe number of color forming units, their size and the intensity of theircolor. TABLE 1 Medium for Pretreatment, Beaming and SelectionIngredients in 1 liter DN62 DN62AG DN62AB DN620SM N6 salts 3.98 g 3.98 g3.98 g 3.98 g N6 vitamins 1 ml 1 ml 1 ml 1 ml Asparagine 800 mg 800 mg800 mg 800 mg Myo-inositol 100 mg 100 mg 100 mg 100 mg Proline 1400 mg1400 mg 1400 mg 1400 mg Casamino acids 100 mg 100 mg 100 mg 100 mg 2,4_D1 mg 1 mg 1 mg 1 mg Sucrose 20 g 20 g 20 g 20 g Glucose 10 g Sorbitol45.5 g Mannitol 45.5 g AgNO₃ 10 mg 10 mg Bialaphos 1 mg Gelrite 3 g 3 g3 g 3 g pH 5.8 5.8 5.8 5.8

Example 3 Introduction of Nucleic Acid into Immature Corn Embryos andDetection of Transient Expression

[0132] Immature embryos of Stine inbred 963 (10 days' post-pollination)were also used for transient expression experiments. In this caseembryos were dissected out at between 1 mm and 2 mm in length and eitherused immediately or maintained for up to 10 days, preferably 2 to 3days, on DN62AG medium (Table 1) prior to beaming. Forty-five minutesbefore beaming the embryos were transferred to DN62OSM. After beaming asdescribed in Example 2 for callus tissue, the embryos were allowed toremain on DN62OSM for 30 minutes before final transfer to DN62AG for oneday.

[0133] One day after treating immature embryos with the aerosol beamapparatus transient expression was evaluated by histochemical analysis.Immature embryos were incubated in the presence of the substrate X-gluc(Gold Biotechnology) at a concentration of 0.5 mg/ml in 0.1 M sodiumphosphate buffer pH 7.0 and 0.1% Triton-x-100 at 37° C. After 1-4 hoursblue spots appeared indicating GUS expression which verifies that theGUS expression cassette was introduced into the cell with the aerosolbeam apparatus. Transient expression results obtained using a DuPont PDS1000 particle delivery device were compared with those obtained usingthe method of the invention. Equivalent amounts of DNA were used. Bluespots appeared more rapidly, were more numerous, larger and more intenseusing the method of the invention.

Example 4 Introduction of Nucleic Acid into Corn Callus and Detection ofStable Transformation

[0134] The plasmids used for stable transformation preferably containedthe bar expression cassette from pBARGUS, as described previously. Thiscassette allowed for selection of a stable transformants using theherbicide bialaphos. Plasmid pRBTBAR contained a Bt expression cassettealong with the bar expression cassette from pBARGUS. Plasmid pBARGFPcontained the bar expression cassette along with a gfp expressioncassette. In the case of this plasmid the bar expression cassette wasfrom pSLJ2011 which was obtained from The Sainsbury Laboratory, England.The bar gene was driven by the CaMV 35S promoter, TMV omega enhancer,and terminated by the octopine synthase terminator (ocs). The gfp (EGFP)gene was obtained from Clonetech, Inc., and was driven by a CaMV 35Spromoter and terminated with a CaMV 35S terminator. For stabletransformation experiments, the DNA concentration varied from 2 ug/ml to20 ug/ml and the DNA was either supercoiled or linear. The supercoiledDNA was isolated as previously described (Example 2). The linear barexpression cassette was isolated by digesting 20 ug of pBARGUS withHindIII as described by the manufacturer Promega, separated on a 1%agarose gel as is common in the art and extracted from the agarose gelusing Qiaex II as described by the manufacturer (Qiagen). Usually 50% ofthe DNA found in a band was recovered which would have been theequivalent in moles to 10 ug pBARGUS.

[0135] Embryogenic callus, maintained on DN62, is transferred off DN62 1to 10 days, preferably 3 to 6 days, after the previous transfer andplaced on DN62OSM for 45 minutes prior to beaming. After beaming asdescribed in Example 1, callus was allowed to remain on this medium for30 minutes. The beamed tissue was then transferred to DN62B (seeTable 1) for selection. Various selection schemes were tested and weresuccessful including selection on bialaphos at concentrations of up to10 mg/l. Success in selection was enhanced by the use of the gfp gene incombination with the bar gene, allowing for visual selection offluorescing transformed tissues.

Example 5 Regeneration of Stably Transformed Corn Callus

[0136] A preferred pathway used to produce stably transformed plantsfrom beamed embryogenic callus (Stine 963) was performed as follows:after beaming on DN62OSM, the callus was transferred to DN62B andcultured for a passage of 14 days; after this period it was transferredagain to further passages of fresh DN62B medium; after between three andsix passages on DN62B clones were identified by growth in the presenceof bialaphos (Table 1).

[0137] Clones were induced to regenerate plants by selecting activelygrowing Type II callus from clonal tissue, with the objective ofobtaining a high frequency of so-called “water tower” embryo structures(U.S. Ser. No. 09/203,679 incorporated herein). This type of tissue ispreferred because regeneration of whole plants is improved. Thesetissues were then transferred to DNROB (Table 2). On this medium embryomaturation occurs. Maturing tissues were then transferred off DNROBafter two to three weeks either to a fresh plate of DNROB or to 0.1NABA6S (Table 2). After a further one to two weeks, embryos with a shootmeristem were placed on MSOG medium (Table 2) or ½MSIBA (Table 2), wheregermination occurs. Plantlets were then transferred to tubes containing½MSIBA medium for promotion of further root and shoot development priorto final transfer to soil. TABLE 2 Media for Regeneration Ingredients in1 liter DNROB 0.1NABA6S MSOG 1/2MSIBA MS Salts 4.43 g 4.43 g 4.43 g2.215 g Asparagine 800 mg Proline 1400 mg Na2EDTA 37.3 mg 37.3 mg 37.3mg 37.3 mg Casamino acids 100 mg Nicotinic Acid 0.5 mg1-naphthaleneacetic 0.1 mg acid Abscisic acid 0.1 mg Gibberellic Acid0.1 mg Indole 3 Butyric 0.1 mg Acid Sucrose 60 g 30 g 20 g Sorbitol 20 gBialaphos 1 mg Gelrite 2 g Phytagar 7 g 7 g 7 g pH 5.8 5.8 5.8 5.8

[0138] The presence of an expressing bar gene was confirmed in theregenerants by leaf painting with Liberty. Mendelian ratios of anexpressing bar gene were routinely observed in the progeny of theregenerants (Table 3). TABLE 3 Target Liberty Liberty Tissue ResistantSensitive Ratio Regenerant* 2 - Event B1 Callus 77 23 3:1 Regenerant 8 -Event B2 Embryo 60 18 3:1 Regenerant 2 - Event B3 Embryo 74 25 3:1Regenerant 1 - Event B5 Callus 71 26 3:1 Regenerant 7 - Event B6 Callus58 41 1.4:1

Example 6 Introduction of Nucleic Acid into Immature Corn Embryos andDetection of Stable Transformation

[0139] Immature embryos of Stine 963 were excised from kernels usuallyaround 10 days' post-pollination. At this time the embryos were around1.0 to 1.5 mm in length. Embryos were then placed on DN62AG medium fortwo to three days. After this time they were then placed on DN62OSM for45 minutes prior to beaming. After beaming as described in Example 1,the embryos remained on DN62OSM for 30 minutes and then were transferredeither to DN62AG for five days prior to transfer to DN62AGB, or toDN62AGB directly (Table 1).

Example 7 Regeneration of Stably Transformed Corn Embryos

[0140] After a further week's culture the corn embryos from Example 6were then transferred to DN62AB (Table 1). After several 14-day passageson DN62AB, clones can then be selected. Clonal tissue was induced toregenerate plants according to the description in Example 5.

[0141] In one experiment 44 immature embryos were beamed and 5 cloneswere recovered (11.3%) which gave rise to transformed plants. Thepresence of an expressing bar gene was confirmed in these clones asdescribed in Example 4.

[0142] Segregation of expression of the bar gene in Stine elite inbred963 after transformation using the method of the invention is shown inTable 3. All regenerants were selfed. Heritability of the nucleic acidinserted by the method of the intention was reflected in the expectedratios for inheritance of a single dominant gene in the progeny oftransformed plants. The fertility of the transgenic plants producedusing the method of the invention is comparable to that ofnontransformed regenerated plants of the same inbred.

[0143] Transformation frequency using immature embryos of Stine eliteinbred 963 and the DuPont PDS-1000 particle gun was around 1% (afterbombardment of several thousand embryos). With the method of the presentinvention, success rates of up to 20% were noted with an average ofaround 3% over all experiments.

Example 8 Improved Growth Rate of Soybean Embryogenic Callus

[0144] In another embodiment of the present invention, a novel culturemedia may be used to stimulate high frequency production of embryogenicsoybean callus. Improvement varied with the genotype being cultured. Thelength of time required for a culture passage was unexpectedly reducedto two weeks with the use of this novel medium as compared to four weekstypical with other media. The inclusion of one or more of four mediaconstituents, coconut water, myoinositol, phytic acid and inorganicphosphate concentration, enhanced embryogenic callus production andallowed significant improvements to be made to transgenic cloneproduction in terms of number of clones recovered, embryo morphology,and reduction in the time needed to identify the clones and regenerateplants from them. The medium of Ranch et al., 1985 (referred to hereinas B1-30) was used as the basal medium. An example of the medium of thepresent invention is B1-30 3Co5My0.25PA0.5K (footnote, Table 5).Although this medium is a preferred emobodiment of growth medium, otherconventional media may be utilized in the practice of the invention.

[0145] Coconut water has been included in tissue culture media for over50 years. Coconut water is liquid taken from coconuts to promote growthin plant tissue cultures. It is deproteinized then filter-sterilized. Anexample is Sigma Biosciences' Cat. No. C5915. The beneficial effects ofcoconut water were first noted by Overbeek et al., 1941, when it wasfound to permit the growth in culture of heart-stage Datura embryos. Insubsequent years, Steward and others showed that coconut water wascapable of stimulating responses from a variety of plant tissues(Steward et al., 1969). Most commonly coconut water is used atconcentrations of between 5% and 10% by volume of the final culturemedium. The role of coconut water in stimulating the growth ofembryogenic callus in soybean was investigated and it was discoveredthat its effect on cotyledon explants routinely used to initiateembryogenic callus of soybean was detrimental when tested at theseconcentrations. No embryogenic callus was produced in these experiments.However, when coconut water was added to media used for the maintenanceof embryogenic callus, an unexpected beneficial effect was noted interms of rate of growth and quality of somatic embryo morphology. Inview of this result, coconut water was routinely included in media forthe maintenance of embryogenic callus at concentrations of between 3%and 6% by volume of the final culture medium.

[0146] One key component of coconut water is myoinositol (Pollard etal., 1961). In an attempt to improve the growth of embryogenic soybeancallus with components of coconut water, it was discovered thatmyoinositol was effective in this regard. Myoinositol has been includedin tissue culture media for a variety of plant species. Althoughapparently not required for all species (Halperin, 1966), it isroutinely included in media such as the widely used Murashige and Skoogformulation (Murashige and Skoog, 1962) at a concentration of 100 mg/l.It is at this concentration that it has been included in media used forthe culture of embryogenic callus of soybean (e.g., see Ranch et al.,1985). Unexpected results revealed a pronounced and beneficial effect ofmyoinositol when used at much higher concentrations (up to 10 g/l) aloneor in combination with coconut water.

[0147] There are no known reports of the inclusion of phytic acid inculture media for any plant species. Soybean seeds are rich in naturalchelating agents, the most prominent of which is phytic acid (Gibson andUllah, 1990). Substantial amounts of phosphate are stored in seeds inthe form of phytate (Raboy, 1990). It is possible that the capacity ofphytic acid to sequester inorganic phosphate has a significant impactupon C partitioning into either starch or sucrose. At 1 gm/l in B1-30,embryogenic callus of a range of genotypes exuded starch, possiblyconfirming the impact of phytic acid on C partitioning. Surprisingly,after this initial effect the soybean callus grew very vigorously andproduced many small globular embryos. Further experiments indicated thatphytic acid at 1, 5, 10, 50, 100, 250, 500 and 1000 mg/l in B1-30significantly increased the rate of growth of embryogenic callus duringthe initial culture passage and/or during maintenance as a stockculture. At 3000 mg/l a clear detrimental effect was observed andembryogenic callus browned and died. Best results over many passageswere obtained with the addition of about 5 mg/l to about 250 mg/l phyticacid to the culture medium depending on the genotype.

[0148] Inorganic phosphorous in the form of KH₂PO₄, in excess of theamount conventionally used, was added to the base medium (B1-30) alongwith myoinositol and coconut water. This medium was tested against B1-30supplemented with phytic acid in addition to KH₂PO₄, myoinositol andcoconut water. Beneficial effects were noted with 500 and 1000 mg/l bothwith and without phytic acid. TABLE 4 Growth Media for Soybean*Ingredients in 1 Liter B1-30 B3 B5G Ms Salts 4.43 g 4.43 g B5 Salts 3.19g NaEDTA 37.3 mg 37.3 mg 37.3 mg 2,4-D 30 mg Activated 5 g CharcoalPhytagar 8 g 8 g Gelrite 2 g pH 5.8 5.8 5.8 # additional KH₂PO₄ and ½B5G which contained all ingredients of B5G medium at half strength.

[0149] Using the improvements described above, new and effective mediawere developed for the production of embryogenic soybean callus from avariety of elite genotypes. (See Example 9). The media of the presentinvention are effective for a range of germplasm and also greatly reducethe time required to obtain embryogenic callus in sufficient quantityfor transformation experiments.

Example 9 Culture of Soybean Callus

[0150] To initiate cultures, pods were surface sterilized and embryoswith immature cotyledons 3 to 4 mm in length were excised. Individualcotyledons were then cultured on B1-30 medium (Table 4) or on B1-30media containing 100 to 1000 mg/l phytic acid. Typically, a small amountof embryogenic callus proliferated from some of these cotyledonexplants. This callus was then excised and transferred to a B1-30 mediumcontaining between 3% and 6% coconut water or/and between 1 g/l and 10g/l myoinositol. The coconut water and myoinositol requirements foroptimum sustained embryogenic callus growth were found to vary accordingto genotype. Exemplary results using a range of concentrations of thesecomponents are summarized in Table 5. TABLE 5 Comparative GrowthResponse Stine Genotype Media Response 96E750 B1-30 3Co 10My best 96E750B1-30 3Co 5My good 96E750 B1-30 3Co good 96E750 B1-30 worst 96E692 B1-303Co 5My best 96E692 B1-30 6Co 5My v. good 96E692 B1-30 3Co 3My v. good96E692 B1-30 3Co 1My good 96E692 B1-30 3Co good 96E692 B1-30 3Co 10Myfair 96E692 B1-30 worst 98CB371 B1-30 3Co 10My best 98CB371 B1-30 3Co5My good 98CB371 B1-30 worst 98CB166 B1-30 3Co 10My best 98CB166 B1-303Co 5My good 98CB166 B1-30 worst

[0151] In other experiments it was unexpectedly discovered that furtherenhancement of embryogenic callus formation was possible by addition ofabout 1 to about 1000 mg/l of phytic acid and/or additional inorganicphosphate in the form of KH₂PO₄ to B1-30 with myoinositol and coconutwater. The amounts required for improvement vary with genotype. Withthese two additional constituents, suitable amounts of embryogeniccallus for transformation experiments can be obtained within three tosix months, a significant improvement over the year or 18 monthstypically required for elite genotypes on standard media, such as B1-30.

[0152] Also effective in preparing tissue for beaming is a briefexposure to coconut water, myoinositol and about 1 gm/l phytic acid forfrom about 3 to 10 days immediately before beaming. Embryogenic callustaken off this medium and beamed directly grows vigorously after beamingin the period before exposure to a selection agent such as bialaphos(Example 11) allowing for greater survival and growth of transformedcells. This improved survival increases the chances of cloneidentification during selection.

Example 10 Introduction of Nucleic Acid into Soybean Callus andDetection of Transient Expression

[0153] The apparatus, parameters and beaming method used to introducenucleic acid into soybean callus were essentially as described inExample 1, unless otherwise indicated in this Example. The samplesolution containing DNA was prepared as previously described (Example2).

[0154] Embryogenic soybean callus of Stine 13404-TT was transferredafter a culture passage of about 28 to 30 days from stock culture medium(B1-30 3Co5My 50 mg/l phytic acid—Table 4) to the center of a targetplate containing the same medium. Embryogenic soybean callus can survivebeing held in a vacuum for at least 10 minutes. After one to three days'growth on the target plate, the soybean embryogenic callus is exposed toan aerosol beam of pSLJ4K1 (the 35 S promoter driving the gus gene).After beaming the tissue is spread out on a fresh plate (to minimize therisk of contamination) of the same medium.

[0155] Approximately one day after treating the soybean callus with theaerosol beam apparatus, transient expression was evaluated byhistochemical analysis. Embryogenic callus was incubated in the presenceof the substrate X-gluc (Gold Biotechnology, Inc.) at a concentration of0.5 mg/ml in 0.1 M sodium phosphate buffer pH 7.0 and 0.1% Triton-x-100at 37° C. After 1-4 hours blue spots appeared indicating GUS expressionwhich verifies that the GUS expression cassette was introduced into thecell with the aerosol beam apparatus. Transient expression results wereused to compare the efficiency of DNA delivery, using equivalent amountsof DNA, to embryogenic callus using either the method of the inventionor a DuPont PDS 1000 particle delivery device. Delivery using the methodof the invention was readily observed to be more efficient both in termsof the number of color forming units and the intensity of their color.

Example 11 Introduction of Nucleic Acid into Soybean Embryogenic Callusand Detection of Stable Transformation

[0156] The plasmids used to stably transform soybean were pSB12BARAHASand pNPTAHAS. Plasmid SB12BARAHAS contained the bar expression cassettefrom pSLJ2011 which consisted of a CaMV 35S promoter, TMV omegaenhancer, bar gene, and nos terminator. Plasmid SLJ2011 was obtainedfrom The Sainsbury Laboratory at The John Innes Center, England. The barexpression cassette was combined with a genomic fragment fromArabidopsis harboring a mutant ahas gene (American Cyanamid). PlasmidpNPTAHAS contained an npt (neomycin phosphotransferase) expressioncassette consisting of a CaMV 35S promoter, TMV omega enhancer, nptgene, and ocs terminator, and the same ahas expression cassettedescribed above. The npt expression cassette was from plasmid SLJ481(Sainsbury Laboratory, John Innes Center, England). The npt expressioncassette provides tolerance to the antibiotic kanamycin and the barexpression cassette provides tolerance to the herbicide bialaphos. Forpurposes of example, supercoiled DNA was used, however, linear DNA ornucleic acid can be expected to work equally well. DNA concentrationvaried from 2 ug/ml to 20 ug/ml. For pSB12BARAHAS approximately 64billion molecules of DNA were delivered per 1 ug.

[0157] Embryogenic calluses of several Stine elite varieties, including96E750, 96E94, 97E986, 96E144 and 96E692, were separately collected intothe center of plates of B1-30 3Co5My or B1-30 3Co5My0.25PA0.5K (Table 4)three days after transfer to fresh medium. The tissue was then beamedwith one of the plasmids described above. After beaming (as described inExample 10) the embryogenic callus was transferred to fresh B1-30 3Co5Myor B1-30 3Co5My0.25PA0.5K for one passage of a month. The tissue wasthen transferred to selective medium containing either 300 mg/lkanamycin or 1 mg/l bialaphos depending on the plasmid used. Withbialaphos, selection typically was maintained at 1 mg/l for the firsttwo one-month passages and then increased to 2 mg/l for the followingthree to seven months. On both bialaphos and kanamycin clones wereidentified after between five and nine transfers on selective medium.Clones were identified when tissue generated by transformationexperiments began to grow vigorously on medium containing a selectiveagent. Once identified, clonal tissue was allowed to increase and wasthen regenerated into plants according to the following protocol: (1)Embryogenic structures were transferred off B1-30 3Co5My or B1-303Co5My0.25PA0.5K to B3 medium (Table 4); (2) after 3 to 4 weeks' growthon this medium clusters of maturing embryos were separated out intoindividual structures and either maintained on the same plate ortransferred to fresh medium; (3) after another 3 to 4 weeks maturingembryos were transferred to B5G medium (Table 4) containing activatedcharcoal and placed in the light; (4) embryos which then elongated andproduced roots were transferred to tubes containing ½ B5G medium (Table4) with no activated charcoal where they continued development intoplantlets; and (5) these plantlets were removed from the tubes andplaced into pots.

[0158] Transformation frequency with embryogenic callus of elite Stinesoybean lines was greater using the method of the invention than thatachieved in similar experiments using the Dupont particle gun. Thisresult was consistent with the results obtained for transient expressionin embryogenic soybean callus where grater delivery of DNA was observedusing the method of the invention when compared to the Dupont gun. Thepresence of an expressing bar gene or an expressing ahas gene wasconfirmed by leaf painting (bar) or spraying (ahas). Mendelian ratios ofthe expressing genes were routinely observed in the progeny of theregenerants.

Example 12 Transformation of Bacteria

[0159] Transformation of bacteria using the method of the invention wasaccomplished as follows: competent E. coli DH5 alpha (Gibco BRL) orHB101 cells were thawed on ice and 5 to 50 microliters were combinedwith 0.2 ml LB broth. The mixture was pipetted to the center of a plateof LB agar (1.5%) containing 100 mg/l ampicillin and allowed to dry.Aerosol beam parameters were the same as those described previously(Example 1) except as noted in this Example. A pUC-derived plasmidencoding for resistance to the antibiotic ampicillin was delivered tothe bacterial cells, as is known in the art. The concentration of DNAranged from about 1.0 to 1000 μg/ml in 10 mM Tris (pH 7.0) or acarbohydrate molecule, such as 1 g/l sucrose. The nebulizing pressurewas from about 10 to about 40 psi, preferably 30 to 40 psi. Theentrainment temperature was maintained in a given experiment and rangedbetween experiments from about 24° to about 55° C., and was preferablymaintained at about 42° to about 55° C. and most preferably at about 55°C. Various sample flow rates were tested ranging from about 0.25 ml/hourto about 2.0 ml/hour (about 4 μl/min. to about 33 μl/min.). Preferablythe flow rate was about 8.0 μl/minute to about 17.0 μl/minute.

[0160] The stage on which the bacterial cells were placed was located ata distance of from about 1.5 cm to about 5.0 cm from the end of thenozzle with a preferred distance of between about 1.5 cm and about 3.2cm. The speed at which the stage moved during beaming was adjusted toabout 10 mm/sec to about 100 mm/sec with a preferred speed of about 20mm/sec to about 100 mm/sec. The distance between passes was varied fromabout 0.2 mm to about 1.0 mm. Most preferable distance between passeswas about 0.3 mm to about 0.4 mm.

[0161] The vacuum in the sample chamber was varied from about 26 psi toabout 30 psi with best results obtained at the higher vacuum pressures,for example 29 psi. Nozzles with orifices of about 254 to 500 micronswere used. Preferable were nozzles with diameters of about 300 to about330 microns.

[0162] After beaming, plates were incubated at 37° C. for 15 to 20hours. No growth was observed in the control without DNA, however, whenDNA was introduced, growth of thousands of transformed colonies wasevident. As a control, the same sample of DNA which was beamed wassprayed on the cells using the same microflow nebulizer used with theaerosol beam apparatus but with no supersonic acceleration of aerosoldroplets. No transformed colonies resulted from this treatment. As anadditional control, bacteria were bombarded with tungsten particlescoated with the same pUC-derived plasmid using the particle gun and aprotocol essentially as described by Smith et al. (1992). This protocolproduced no transformants. The protocol of Smith et al. (1992), wasaltered in the practice of the present invention in that the relativehumidity was not adjusted nor was osmoticum used.

Example 13 Use of the Method of the Invention to Increase TransformationFrequency

[0163] Transformation frequency in plants can be increased byintroducing restriction enzymes simultaneously with the transforming DNAusing the method of the present invention. Parameters for the aerosolbeam apparatus are as described previously for plant cells (Example 1).Corn and soybean target tissue are prepared as described previously(Examples 4 and 5). For selection of transformants, a number ofdifferent selective agents can be used including, but not limited to,bialaphos, kanamycin, hygromycin, and imazapyr. By way of illustration,selection using imazapyr and mutant AHAS genes is described.

[0164] Plasmid pCD220 carries a mutant corn AHAS gene. Expression ofthis gene in corn confers resistance to the herbicide Arsenal (activeingredient imazapyr) manufactured by American Cyanimid. The plasmid isdigested with the restriction enzyme Xba I as described by Promega. Thelinearized DNA fragment carrying the gene and regulatory elementsnecessary for expression in corn are separated from the remainingplasmid DNA in a 1% agarose gel as practiced in the art. The genomic DNAis then purified from the gel using the QIAEX II gel extraction kitaccording to the manufacture (Qiagen). About 10 micrograms of linear DNAwith Xba I compatible ends and 200 units of Xba enzyme are combined in 1ml of 10 mM Tris pH 7.0, or in 1 ml of a buffer solution recommended forXba I by the manufacturer (Promega). The solution containing the DNA andthe enzyme is then beamed into corn cells as previously described.Transformants are selected as described previously. The simultaneousintroduction of DNA with the appropriate restriction enzyme cansignificantly increase the frequency of transformation.

[0165] A mutant AHAS gene from Arabidopsis can be introduced intosoybean cells using the same methods. Plasmid AC321 is digested with XbaI as described above for plasmid pCD220. This Arabidopsis genomicfragment is then used for transformation of soybean together with theXba I restriction enzyme. Again, the number of transformants obtainedusing the enzyme together with the DNA can be significantly higher thanwhen using the DNA alone.

Example 14 Introduction of Carbohydrate and Plant Growth Regulator intoCells

[0166] Glucose and 2,4-D were introduced into cells of cultured immatureembryos of Stine corn inbred 963 by the method of the present invention.Preparation of embryos and parameters for the aerosol beam apparatuswere the same as described for Example 6. Glucose was used at aconcentration of 1 gm/l. 2,4-D was used at a concentration of 0.1 mg/l.After beaming with pBARGUS, glucose, and 2,4-D, stably transformedclonal tissue was selected and plants regenerated as described inExample 7. A significant increase (2 to 10 fold) in the number of clonesrecovered was noted from embryos beamed with glucose, 2,4-D and DNA whencompared with embryos beamed with DNA alone. Similar results wereobtained using 1 g/l sucrose in place of 1 g/l glucose.

Example 15 Introduction of Protein into Cells

[0167] The protein glucuronidase was introduced into embryogenic calluscells of Stine corn inbred 963 by the method of the present invention.Target preparation and aerosol beam parameters were as described inExample 2. Glucuronidase from Sigma (cat # G2035) was used at aconcentration of 320 μg/ml in 10 mM Tris (pH 7.0). After treating ofcorn callus with the aerosol beam apparatus, the tissue was immediatelyincubated in X-gluc substrate, as described in Example 2. After 1 to 4hours blue spots appeared indicating the intracellular presence of GUS.The intracellular presence of GUS was then confirmed by microscopicexamination. As a control, protein was applied to the surface of theembryogenic callus with an atomizer and the callus was then incubatedfor about 1 to 4 hours in x-gluc substrate. No blue spots were observedwith this treatment.

Example 16 Introduction of Nucleic Acid together with Other Moleculesinto Cells

[0168] Nucleic acid and protein were simultaneously delivered intobacterial cells. Preparation of bacteria and parameters for the aerosolbeam apparatus were the same as described previously for delivery of DNAinto bacteria (Example 12). The same buffer, plasmid, and plasmidconcentration was used as described in the transformation of bacteria.Ribonuclease A (RNASE A) (Sigma cat. no. R6513) was added to the DNAsample at a final concentration of 2.5 μg/ml. After the sample of DNAand RNASE A was beamed into bacteria, no colonies were observed onmedium containing ampicillin indicating the cytotoxic effect of RNASE Ain the cells.

[0169] As a control, RNASE A at the same concentration used for beamingwas pipetted on the cells after the cells were beamed with DNA only.Here, transformed colonies grew indicating that the RNASE A was nottaken up naturally by the cell. To verify that the RNASE A was notdegrading the plasmid, the plasmid treated with RNASE A was run on astandard 1% agarose gel. After staining the DNA with ethidium bromide,the DNA appeared intact and no degradation was evident. As a furthercontrol, RNASE A was inactivated with diethylpyrocarbonate (DEP).Inactivation was carried out as follows: 2.5 micrograms of RNASE A wasadded to 1.0 ml 10 mM Tris (pH 7.0), then 4.0 μl of DEP was added. Thismixture was vortexed and incubated for 2-4 hours at room temperature,after which time, gases were evacuated from the tube by heating themixture to 85° C. for 15 minutes with the cap of the tube off. DNA wasthen added and the sample beamed into cells. The introduction of DNA andinactivated RNASE A resulted in the normal transformation of bacteria.Thus, the addition of RNASE A to the DNA did not prevent the DNA fromentering the cell. From these experiments it follows that both DNA andprotein were delivered together into bacterial cells.

Example 17 Optimization of Sample Flow Rate for Bacteria

[0170] Transformation of bacteria using the method of the invention wasperformed as described in Example 12. The sample flow rate was variedfrom about 1 μl/minute to about 200 μl/minute. Although sucrose was usedin this example, other carbohydrates such as glucose can be used in thepractice of the invention. The efficiency of DNA delivery is reflectedin Table 6 as number of bacterial colonies able to grow on agarcontaining 100 mg/l ampicillin at the listed sample flow rates.

[0171] The preferred sample flow rate for introduction of DNA intobacteria was about 1 μl/minute to about 200 l/minute, a more preferredflow rate was about 4 μl/minute to about 50 μl/minute and the mostpreferred flow rate was about 8 μl/minute to about 17 μl/minute.

Example 18 Optimization of Sample Flow Rate for Corn

[0172] Introduction of DNA into immature corn embryos was performedusing the method of the invention as described in Example 3. The sampleflow rate was varied from about 1 l/minute to about 200 μl/minute.Transient expression results were used to compare the efficiency of DNAdelivery. Results are reflected in Table 6 as number of blue spots perembryo at the listed sample flow rates. The preferred sample flow ratefor introduction of DNA into corn is about 1 μl/minute to 200 μl/minute,a more preferred rate is about 4 μl/minute to about 50 μl/minute and amost preferred rate is about 8 μl/minute to about 50 μl/minute.

Example 19 Optimization of Sample Flow Rate for Soybean

[0173] Introduction of DNA into soybean callus was performed using themethod of the invention as described in Example 10. The sample flowrates varied from about 1 μl/minute to about 200 μl/minute. Transientexpression results were used to compare the efficiency of DNA delivery.Results are reflected in Table 6 as number of blue spots per plate atthe listed sample flow rates. The preferred sample flow rate forintroduction of nucleic acid into soybean callus was about 1 μl/minuteto about 200 μl/minute, with a more preferred rate of about 4 μl/minuteto about 50 μl/minute and a most preferred rate of about 8 μl/minute toabout 50 μl/minute. TABLE 6 Flow Rate in Microliters Per Minute* 1.0 2.04.0 8.0 17.0 50.0 100.0 200.0 Bacteria 0 0 24 236 179 91 2 2 Corn <1.01.0 4.0 7.5 9.0 8.0 1.0 <1.0 Soybean 0 0 139 264 249 211 65 22 # by thenumber of blue spots per embryo, and for soybean the number of bluespots are per plate.

[0174] While the invention has been disclosed in this patent applicationby reference to the details of preferred embodiments of the invention,it is to be understood that the disclosure is intended in anillustrative rather than a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of appended claims.

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[0232] Patents and Patent Applications:

[0233] Zhao, Z. Y., et al., published PCT WO 98/32326 published in 1988.

[0234] U.S. Pat. No. 4,743,548

[0235] U.S. Pat. No. 5,240,842

[0236] U.S. Pat. No. 5,384,253

[0237] U.S. Pat. No. 5,472,869

[0238] U.S. Pat. No. 5,550,318

[0239] U.S. Pat. No. 5,591,616

[0240] U.S. Pat. No. 5,877,023

1. A method for the introduction of one or more peptides into a cell,wherein the method comprises: (a) preparing a solution containing saidpeptides; (b) supplying said solution for conversion to aerosol dropletswhich are produced by a microflow nebulizer at a flow rate of betweenabout 1 μl/minute to about 100 μl/minute; (c) producing aerosol dropletscomprising said peptides; (d) accelerating said aerosol droplets towardsaid cell; (e) impacting said cell with said accelerated aerosoldroplets; and (f) producing a cell containing said peptides. 2.-7.(CANCELED)
 8. The method of claim 1, wherein said cell is a plant cell.9. The method of claim 8, wherein said plant cell is a monocotyledonousplant cell.
 10. The method of claim 9, wherein said monocotyledonousplant cell is a corn cell.
 11. The method of claim 8, wherein said plantcell is a dicotyledonous plant cell.
 12. The method of claim 11, whereinsaid dicotyledonous cell is a soybean cell.
 13. (CANCELED)
 14. Themethod of claim 1, wherein said aerosol droplets are continuouslytargeted toward said cell.
 15. The method of claim 1, further comprisingthe placement of said cell on a target surface the linear and rotationalmovement of which can be separately controlled. 16.-18. (CANCELED) 19.The method of claim 8, wherein said method further comprisesregenerating a plant containing said peptides. 20.-55. (CANCELED) 56.The method of claim 10, wherein the said flow rate is between about 4μl/minute and about 50 μl/minute.
 57. The method of claim 56, whereinthe said flow rate is between about 8 μl/minute and about 50 μl/minute.58. (CANCELED)
 59. The method of claim 12, wherein the said flow rate isbetween about 4 μl/minute and about 50 μl/minute.
 60. The method ofclaim 59, wherein the said flow rate is between about 8 μl/minute andabout 50 μl/minute. 61.-63. (CANCELED)
 64. The method of claim 8,wherein the said flow rate is between about 4 μl/minute and about 50μl/minute.
 65. The method of claim 64, wherein the said flow rate isbetween about 8 μl/minute and about 50 μl/minute.